Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase

Abstract

The mycobacterial cell wall core consists of an outer lipid layer of mycolic acids connected, via arabinogalactan polysaccharide, to an inner peptidoglycan layer. An a-L-rhamnopyranosyl residue has been shown to be a key component USA linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibitors of its biosynthesis. Biochemical assays were used to show that dTDP-Rha was synthesized in Mycobacterium smegmatis from a-D-glucose I-phosphate (a-D-Glc-1-P) and dTTP by the same four enzymic steps used by Escherichia coli and other bacteria. PCR primers based on consensus regions of known sequences of the first enzyme in this series, E-D-GIC-I-P thymidylyltransferase (RfbA) were used to amplify rfbA DNA from M. tuberculosis. The entire rfbA gene was then cloned and sequenced. The deduced amino acid sequence revealed a 31 362 Da putative protein product which showed similarity to RfbA proteins of other bacteria (59% identity to that found in E. coli). Sequencing of DNA flanking the rfbA gene did not reveal any of the other rfb genes required for dTDP-Rha biosynthesis. Therefore, the four Rha biosynthetic genes are not clustered in M. tuberculosis. The enzymic activity of the sequenced gene product was confirmed by transformation of E. coli with pBluescript KS(-) containing the rfbA gene from M. tuberculosis. Analysis of enzyme extracts prepared from this transformant revealed an 1 I-fold increase in E-D-GIc-I-P thymidylyltransferase activity.

Modification of glycopeptidolipids by an O-methyltransferase of Mycobacterium smegmatis a aThe GenBank accession number for the sequence determined in this work is AY138899.

“…In all mycobacteria, including non-GPL-expressing species, rhamnose biosynthesis is of interest because this residue links the arabinogalactan portion of the cell wall core to peptidoglycan (McNeil et al, 1990). Rhamnose biosynthesis has been delineated in M. tuberculosis and M. smegmatis and genes encoding the key enzymes have been characterized in M. tuberculosis (Cole et al, 1998 ;Ma et al, 1997). Unlike the situation in enteric bacteria the mycobacterial genes for rhamnose biosynthesis are dispersed around the chromosome in a similar fashion to that described in Saccharopolyspora spinosa (Madduri et al, 2001).…”

“…Two genes in the GPL locus possibly encode the enzymes for the first two steps of rhamnose biosynthesis. The rmlA gene product is 92 % similar to RmlA of M. tuberculosis, which encodes glucose-1-phosphate thymidylyltransferase and has been shown to convert glucose 1-phosphate to dTDPglucose (Liu & Thorson, 1994 ;Ma et al, 1997). Several glucose-1-phosphate thymidylyltransferases have a conserved motif in the N-terminus which is also observed in the M. smegmatis RmlA polypeptide sequence (LAGGSGTRLHP) (Aguirrezabalaga et al, 2000 ;Merson-Davies et al, 1994 ;Steffensky et al, 2000).…”

“…Genes encoding the four enzymes for rhamnose synthesis (glucose-1-phosphate thymidylyltransferase, dTDP-glucose 4,6-dehydrogenase, dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP dehydrorhamnose reductase) are conserved among bacteria (Stevenson et al, 1994). Rhamnose biosynthesis has been described in M. tuberculosis and M. smegmatis and genes encoding the enzymes above have been identified in M. tuberculosis (Cole et al, 1998 ;Ma et al, 1997). Two genes in the GPL locus possibly encode the enzymes for the first two steps of rhamnose biosynthesis.…”

Conversion of dTDP-4-keto-6-deoxyglucose to free dTDP-4-keto-rhamnose by the rmlC gene products of Escherichia coli and Mycobacterium tuberculosis

“…The genes encoding the first two enzymes in the pathway, rmfA (a-D-glucose-1-phosphate thymidylyltransferase) and rmlB (dTDP-~-glucose-4',6'-dehydratase) have been cloned and expressed from several different organisms (Ma et al, 1997;Lindquist et al, 1993;Romana et af., 1991;Marumo et al, 1992) and shown to catalyse the expected reactions shown in Fig. 1.…”

A Glycosyltransferase Involved in Biosynthesis of Triglycosylated Glycopeptidolipids inMycobacterium smegmatis: Impact on Surface Properties

“…1B). The rmlA and rmlB genes may be involved in rhamnose biosynthesis, according to their sequence homology with rmlA of M. tuberculosis and gepiA of M. avium, respectively (13,25,31). Rv1174c is uncharacterized, and nothing is known about the three putative glycosyltransferases (gtf1, gtf2, and gtf3 genes) present in the GPL locus.…”